Intestinal trefoil proteins

ABSTRACT

Intestinal trefoil factors and nucleic acids encoding intestinal trefoil factors are disclosed. The intestinal trefoil factors disclosed are resistant to destruction in the digestive tract and can be used for the treatment of peptic ulcer diseases, inflammatory bowel diseases and other insults.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a is a continuation of U.S. Ser. No.09/811,259 filed Mar. 16, 2001, which is a continuation of U.S. Ser. No.08/631,469, filed Apr. 12, 1996 (now U.S. Pat. No. 6,221,840), which isa continuation-in-part of U.S. Ser. No. 08/191,352, filed Feb. 2, 1994(abandoned), which is a continuation of U.S. Ser. No. 08/037,741, filedMar. 25, 1993 (abandoned), which is a continuation of U.S. Ser. No.07/837,192, filed Feb. 13, 1992 (abandoned), which is acontinuation-in-part of U.S. Ser. No. 07/655,965, filed Feb. 14, 1991(abandoned).

BACKGROUND

[0002] The field of the invention is peptides useful for treatment ofdisorders of the digestive system.

[0003] Jorgensen et al. (1982, Regulatory Peptides 3:231) describe aporcine pancreatic peptide, pancreatic spasmolytic peptide (PSP). PSPwas found to inhibit “gastrointestinal motility and gastric acidsecretion in laboratory animal after parenteral as well as oraladministration.” It was suggested that “if the results in animalexperiments can be confirmed in man, PSP may possess a potential utilityin treatment of gastroduodenal ulcer diseases.”

SUMMARY OF THE INVENTION

[0004] In a first aspect, the invention features a purified nucleic acidencoding an intestinal trefoil factor (ITF).

[0005] In preferred embodiments, the intestinal trefoil factor ismammalian intestinal trefoil factor, preferably human, rat, bovine, orporcine intestinal trefoil factor. In another preferred embodiment, thepurified nucleic acid encoding an intestinal trefoil factor is presentwithin a vector.

[0006] In a related aspect, the invention features a cell that includesa vector encoding an intestinal trefoil factor.

[0007] In another related aspect, the invention features a substantiallypure intestinal trefoil factor. In a preferred embodiment, thepolypeptide is detectably labeled. In a related aspect, the inventionfeatures a therapeutic composition that includes an intestinal trefoilfactor and a pharmacologically acceptable carrier.

[0008] In another aspect, the invention features a monoclonal antibodywhich preferentially binds (i.e., forms an immune complex with) anintestinal trefoil factor. In a preferred embodiment, the monoclonalantibody is detectably labelled.

[0009] In a related aspect, the invention features a method fordetecting human intestinal trefoil factor in a human patient. The methodincludes the steps of contacting a biological sample obtained from thepatient with a monoclonal antibody which preferentially binds intestinaltrefoil factor, and detecting immune complexes formed with themonoclonal antibody. In preferred embodiments the biological sample isan intestinal mucosal scraping, or serum.

[0010] In a related aspect, the invention features a method for treatingdigestive disorders in a human patient, which method involvesadministering to the patient a therapeutic composition that includes anintestinal trefoil factor and a pharmacologically acceptable carrier.

[0011] In another aspect, the invention features a method for detectingbinding sites for intestinal trefoil factor in a patient. The methodinvolves contacting a biological sample obtained from the patient withthe factor, and detecting the factor bound to the biological sample asan indication of the presence of the binding sites in the sample. By“binding sites,” as used herein, is meant any antibody or receptor thatbinds to an intestinal trefoil factor protein, factor, or analog. Thedetection or quantitation of binding sites may be a useful reflection ofabnormalities of the gastrointestinal tract.

[0012] In another aspect, the invention features substantially puretrefoil factor. In preferred embodiments, the intestinal trefoil factoris human, porcine, or bovine trefoil factor.

[0013] By “intestinal trefoil factor” (“ITF”) is meant any protein thatis substantially homologous to rat intestinal trefoil factor (FIG. 2;SEQ ID NO.: 2) and which is expressed in the large intestine, smallintestine, or colon to a greater extent than it is expressed in tissuesother than the small intestine, large intestine, or colon. Also includedare: allelic variations; natural mutants; induced mutants; proteinsencoded by DNA that hybridizes under high or low stringency conditionsto ITF encoding nucleic acids retrieved from naturally occurringmaterial; and polypeptides or proteins retrieved by antisera to ITF,especially by antisera to the active site or binding domain of ITF. Theterm also includes other chimeric polypeptides that include an ITF.

[0014] The term ITF also includes analogs of naturally occurring ITFpolypeptides. Analogs can differ from naturally occurring ITF by aminoacid sequence differences or by modifications that do not affectsequence, or by both. Analogs of the invention will generally exhibit atleast 70%, more preferably 80%, more preferably 90%, and most preferably95% or even 99%, homology with all or part of a naturally occurring ITFsequence. The length of comparison sequences will generally be at least8 amino acid residues, usually at least 20 amino acid residues, moreusually at least 24 amino acid residues, typically at least 28 aminoacid residues, and preferably more than 35 amino acid residues.Modifications include in vivo, or in vitro chemical derivatization ofpolypeptides, e.g., acetylation, or carboxylation. Also included aremodifications of glycosylation, e.g., those made by modifying theglycosylation patterns of a polypeptide during its synthesis andprocessing or in further processing steps, e.g., by exposing thepolypeptide to enzymes that affect glycosylation derived from cells thatnormally provide such processing, e.g., mammalian glycosylation enzymes.Also embraced are versions of the same primary amino acid sequence thathave phosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine. Analogs can differ from naturallyoccurring ITF by alterations of their primary sequence. These includegenetic variants, both natural and induced. Induced mutants may bederived by various techniques, including random mutagenesis of theencoding nucleic acids using irradiation or exposure toethanemethylsulfate (EMS), or may incorporate changes produced bysite-specific mutagenesis or other techniques of molecular biology. See,Sambrook, Fritsch and Maniatis (1989), Molecular Cloning: A LaboratoryManual (2d ed.), CSH Press, hereby incorporated by reference. Alsoincluded are analogs that include residues other than naturallyoccurring L-amino acids, e.g., D-amino acids or non-naturally occurringor synthetic amino acids, e.g., β or γ amino acids.

[0015] In addition to substantially full-length polypeptides, the termITF, as used herein, includes biologically active fragments of thepolypeptides. As used herein, the term “fragment,” as applied to apolypeptide, will ordinarily be at least 10 contiguous amino acids,typically at least 20 contiguous amino acids, more typically at least 30contiguous amino acids, usually at least 40 contiguous amino acids,preferably at least 50 contiguous amino acids, and most preferably atleast 60 to 80 or more contiguous amino acids in length. Fragments ofITF can be generated by methods known to those skilled in the art. Theability of a candidate fragment to exhibit a biological activity of ITFcan be assessed by methods known to those skilled in the art. Alsoincluded in the term “fragment” are biologically active ITF polypeptidescontaining amino acids that are normally removed during proteinprocessing, including additional amino acids that are not required forthe biological activity of the polypeptide, or including additionalamino acids that result from alternative mRNA splicing or alternativeprotein processing events.

[0016] An ITF polypeptide, fragment, or analog is biologically active ifit exhibits a biological activity of a naturally occurring ITF, e.g.,the ability to alter gastrointestinal motility in a mammal.

[0017] The invention also includes nucleic acid sequences, and purifiedpreparations thereof, that encode the ITF polypeptides described herein.The invention also includes antibodies, preferably monoclonalantibodies, that bind specifically to ITF polypeptides.

[0018] As used herein, the term “substantially pure” describes acompound, e.g., a nucleic acid, a protein, or a polypeptide, e.g., anITF protein or polypeptide, that is substantially free from thecomponents that naturally accompany it. Typically, a compound issubstantially pure when at least 60%, more preferably at least 75%, morepreferably at least 90%, and most preferably at least 99%, of the totalmaterial (by volume, by wet or dry weight, or by mole per cent or molefraction) in a sample is the compound of interest. Purity can bemeasured by any appropriate method, e.g., in the case of polypeptides,by column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

[0019] By “isolated DNA” is meant DNA that is free of the genes which,in the naturally-occurring genome of the organism from which the givenDNA of the invention is derived, flank the DNA. The term “isolated DNA”thus encompasses, for example, cDNA, cloned genomic DNA, and syntheticDNA. A “purified nucleic acid”, as used herein, refers to a nucleic acidsequence that is substantially free of other macromolecule (e.g., othernucleic acids and proteins) with which it naturally occurs within acell. In preferred embodiments, less than 40% (and more preferably lessthan 25%) of the purified nucleic acid preparation consists of suchother macromolecule.

[0020] “Homologous”, as used herein, refers to the subunit sequencesimilarity between two polymeric molecules, e.g., between two nucleicacid molecules, e.g., two DNA molecules, or two polypeptide molecules.When a subunit position in both of the molecules is occupied by the samemonomeric subunit, e.g., if a position in each of two DNA molecules isoccupied by adenine, then they are homologous at that position. Thehomology between two sequences is a direct function of the number ofmatching or homologous positions, e.g., if half, e.g., 5 of 10, of thepositions in two compound sequences are homologous then the twosequences are 50% homologous; if 90% of the positions, e.g., 9 of 10,are matched or homologous the two sequences share 90% homology. By wayof example, the DNA sequences 3′ATTGCC+5 and 3′TATGGC′5 share 50%homology. By “substantially homologous” is meant largely but not whollyhomologous.

[0021] The ITF proteins of the invention are resistant to destruction inthe digestive tract, and can be used for treatment of peptic ulcerdiseases, inflammatory bowel diseases, and for protection of theintestinal tract from injury caused by insults such as radiation injuryor bacterial infection. An ITF protein, fragment, or analog can also beused to treat neoplastic cancer.

[0022] In general, trefoil proteins, including ITF, are useful for thetreatment of disorders of and damage to the alimentary canal, includingthe mouth, esophagus, stomach, and large and small intestine.

[0023] One of the most common bacterial infections is caused byHelicobacter pylori (H. pylori), which leads to active, chronicgastritis and frequently to associated syndromes such as duodenal ulcer,gastric ulcer, gastric cancer, MALT lymphoma, or Menetrier's syndrome.Eradication of H. pylori has been shown to reduce the recurrence ofduodenal and gastric ulcers. Furthermore, it has been postulated thatwidespread treatment of H. pylori will reduce the incidence of gastriccarcinoma, which is the second leading cause of cancer related deathworld-wide.

[0024] Long-standing gastritis associated with H. pylori infection isoften associated with the expression of intestinal-like features in thegastric mucosa. This condition, referred to as intestinal metaplasia(IM), may signal an increased risk of gastric cancer. The etiology of IMis unclear; it could represent a mutational adaptation or defenseagainst H. pylori infection. For example, the metaplastic mucosa mayproduce mucus or other substances that create an environment that ishostile to H. pylori. ITF can be used in the treatment of H. pyloriinfection and conditions associated with H. pylori infection (e.g.,ulcers, gastric carcinoma, non-ulcer dyspepsia, gastritis, andesophageal lesions associated with gastro-esophageal reflux disease).ITF is useful for treatment of these conditions because of its generallyprotective effect on the gastrointestinal tract. In addition, ITFpromotes the maintenance of mucosal integrity. ITF can be used toinhibit adhesion to or colonization of the mucosa by H. pylori. In thisapplication ITF or fragments or variants thereof which inhibit adhesionor colonization of the mucosa by H. pylori are useful. Such moleculescan be identified using assays known to those skilled in the art,including the H. pylori binding assay described below.

[0025] ITF may also be used promote healing of tissues damaged byconditions associated with H. pylori infection. In this regard, it isimportant that addition of trefoil proteins to wounded monolayers ofconfluent intestinal epithelial cells increases the rate of epithelialcell migration into the wound. This effect is enhanced by concomitantaddition of mucin glycoproteins, the other dominant product of gobletcells.

[0026] Just as ITF can be used to protect other parts of thegastro-intestinal tract or alimentary canal, such as the intestine, itcan be used to protect the mouth and esophagus from damage caused byradiation therapy or chemotherapy. ITF can also be used to protectagainst and/or to treat damage caused by alcohols or drugs generally.

[0027] Members of the trefoil family, including ITF, can be used in thetreatments discussed above. Skilled artisans may review these proteinsin Sands et at. (1996, Ann. Rev. Physiol. 58:253-273). As stated above,the invention encompasses biologically active fragments of the trefoilproteins. Fragments that retain the trefoil structure (i.e., the threeloop structure) or that lie within regions of the protein that arehighly conserved may prove particularly useful. Thus, portions of ITFfrom about the first cys involved in a disulfide bond of the three loopstructure to about the last cys involved in a disulfide bond of thethree loop structure.

[0028] Variants of a selected trefoil protein are least 60%, preferablyat least 75%, more preferably at least 90%, and most preferably at least95% identical to the selected trefoil protein, preferably a humantrefoil protein, more preferably human ITF.

[0029] The term “identical,” as used herein in reference to polypeptideor DNA sequences, refers to the subunit sequence identity between twomolecules. In the case of amino acid sequences that are less than 100%identical to a reference sequence, the non-identical positions arepreferably, but not necessarily, conservative substitutions for thereference sequence. Conservative substitutions typically includesubstitutions within the following groups: glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and phenyalanine,tyrosine. Sequence identity is typically measured using sequenceanalysis software such as the Sequence Analysis Software Package of theGenetics Computer Group at the University of Wisconsin (BiotechnologyCenter, 1710 University Avenue, Madison, Wis. 53705), and the defaultparameters specified therein.

[0030] A variant of a selected trefoil protein preferably has the aminoacids present in the naturally-occurring form of the selected trefoilprotein at the more highly conserved amino acid positions of theprotein. Thus, a variant of human ITF preferably is identical tonaturally-occurring human ITF at all or nearly all of the more highlyconserved positions. Sequence conservation among trefoil proteins isevident in Table 1 of Sands et al. (supra) which can be used by thoseskilled in the art to identify more conserved residues.

[0031] The invention features a method for the treatment of lesions inthe alimentary canal of a patient by administering to the patient atleast one trefoil peptide, or a biologically active fragment thereof.The lesions typically occur in the mucosa of the alimentary canal, andmay be present in the mouth, esophagus, stomach, or intestine of thepatient. The lesions can be caused in several ways. For example, thepatient may be receiving radiation therapy or chemotherapy for thetreatment of cancer. These treatments typically cause lesions in themouth and esophagus of the patient. Skilled artisans will recognize thatit may be useful to administer the proteins of the invention to thepatient before such treatment is begun. Alternatively, the lesions canbe caused by: (1) any other drug, including alcohol, that damages thealimentary canal, (2) accidental exposure to radiation or to a causticsubstance, (3) an infection, or (4) a digestive disorder including butnot limited to non-ulcer dyspepsia, gastritis, peptic or duodenal ulcer,gastric cancer, MALT lymphoma, Menetrier's syndrome, gastro-esophagealreflux disease, and Crohn's disease. The peptide that is administeredmay be any peptide in the trefoil family, such as intestinal trefoilpeptide (ITF), spasmolytic peptide (SP), and pS2. For the treatment ofhuman patients it is expected that the peptide will be expressed by ahuman gene. However, eucaryotic trefoil peptides, such as those clonedfrom the rat and mouse genomes may also prove effective. These peptidesmay be isolated from a naturally occurring source or synthesized byrecombinant techniques. It is expected that the typical route ofadministration will be oral. Determining other routes of administration,and the effective dosage are well within the skills of ordinary artisansand will depend on many factors known to these artisans. The trefoilproteins may be administered singly, in combination with one another,and/or in combination with mucin glycoprotein preparations.

[0032] “Treatment of lesions” encompasses both the inhibition of theformation of lesion and the healing of lesions already formed.Biologically active fragments and variants of a trefoil protein,particularly ITF, which promote healing of lesions or inhibit theformation of lesions are useful in the treatments of the invention.

[0033] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034]FIG. 1 is a depiction of the nucleotide sequence of rat trefoilfactor (SEQ ID NO.: 1).

[0035]FIG. 2 is a depiction of the deduced amino acid sequence of rattrefoil factor (SEQ ID NO.: 2).

[0036]FIG. 3 is a depiction of the amino acid sequences of rat trefoilfactor, pS2 protein, and pancreatic spasmolytic polypeptide (SP). Thesequences are aligned to illustrate the amino acid sequence homologybetween the proteins. Dashes (-) indicate the insertion of spaces whichoptimize alignment. Bars indicate sequence identity.

[0037]FIG. 4 depicts the disulfide bond structure proposed for pS2 (SEQID NO.: 15)(panel A) and PSP (SEQ ID NO.: 16)(panel B).

[0038]FIG. 5 is a depiction of the proposed disulfide bond structure ofrat intestinal trefoil factor (SEQ ID NO.: 17).

[0039]FIG. 6 is a depiction of the nucleotide sequence of the humanintestinal trefoil factor cDNA and the corresponding deduced amino acidsequence (SEQ ID NO.: 3).

[0040]FIG. 7 is a diagram depicting the strategy used to mutate the ITFgene in embryonic stem cells.

[0041]FIG. 8 is a graph depicting survival following administration ofDextran Sulfate Sodium (DSS; 2.5% w/v in drinking water for 9consecutive days), shown as Kaplan-Meier transform of probability versusdays of DSS treatment.

DETAILED DESCRIPTION

[0042] Purification and Cloning of rITF

[0043] An inhibitor of soft agar colony formation by human breastcarcinoma-derived BT-20 cells (ATTC HTB79) was isolated fromcytology-positive human malignant effusions (Podolsky et al., 1988,Cancer Res. 48:418; hereby incorporated by reference). The factor alsoinhibited soft agar colony formation by human colon carcinoma-derivedHCT15 cells (ATTC-CCL225). Inhibition was not observed for polyoma andmurine sarcoma virus transformed rodent fibroblast lines. The isolatedfactor (transformed cell-growth inhibiting factor or TGIF) had anapparent molecular weight of 110,000 kD and appeared to consist of two55,000 kD subunits linked by sulfbydryl bonds.

[0044] The purified protein was partially sequenced. The sequence fromthe amino terminal 14 amino acids was used to produce a set ofdegenerate oligonucleotide probes for screening of a rat intestinalepithelial cell cDNA library.

[0045] A rat intestinal cDNA library (Lambda ZAP° II, Stratagene, LaJolla, Calif.) was produced by standard techniques (Ausubel et al.,Eds., Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, 1989) using cells purified by the method of Weisner (1973, J. BiolChem. 248:2536). Screening of the cDNA library with the fully degenerateoligonucleotide probe described above resulted in the selection of 21clones. One of the clones (T3411) included a core sequence which encodeda single open reading frame. The nucleotide sequence of the open readingframe and flanking DNA is presented in FIG. 1 (SEQ ID NO.: 1). Theinsert present in T3411 was nick translated (Ausubel et al., supra) toproduce a radioactively labelled probe for Northern blot analysis of ratpoly(A)⁺ RNA. Northern analysis demonstrated that RNA corresponding tothe cloned cDNA fragment was expressed in small intestine, largeintestine, and kidney; no expression was detected in the lung, spleen,heart, testes, muscle, stomach, pancreas, or liver. In the tissues inwhich the RNA was expressed, the level was comparable to that of actin.

[0046] The open reading frame of clone T3411 encoded an 81 amino acidpeptide (FIG. 2; SEQ ID NO.: 2). Comparison of the sequence of theencoded peptide, referred to as rat intestinal trefoil factor (rITF), tothe sequence of proteins in the Genebank database revealed significanthomology to human breast cancer associated peptide (pS2; Jakowlev etal., 1984, Nucleic Acids Res. 12:2861) and porcine pancreaticspasmolytic peptide (PSP; Thim et al., 1985 Biochem. Biophys. Acta.827:410). FIG. 3 illustrates the homology between rITF, PSP and pS2.Porcine pancreatic spasmolytic factor (PSP) and pS2 are both thought tofold into a characteristic structure referred to as a trefoil. A trefoilstructure consists of three loops formed by three disulfide bonds. pS2is thought to include one trefoil (FIG. 4A), and PSP is thought toinclude two trefoils (FIG. 4B). The region of rITF (nucleotide 114 tonucleotide 230 which encodes cys to phe), which is most similar to PSPand pS2, includes six cysteines all of which are in the same position asthe cysteines which make up the trefoil in pS2 (FIG. 3). Five of thesesix cysteines are in the same position as the cysteines which form theamino terminal trefoil of PSP (FIG. 3). FIG. 5 depicts the proposeddisulfide bond configuration of rITF.

[0047] Based on homology to PSP and pS2 (Mori et al., 1988, Biochem.Biophys. Res. Comm. 155:366; Jakowlew et al., 1984 Nucleic Acids Res.12:2861), rITF includes a presumptive pro-sequence (met1 to ala22) inwhich 12 of 22 amino acids have hydrophobic side chains.

[0048] Production of Anti-rITF Antibodies

[0049] A peptide corresponding to the carboxy-terminal 21 amino acids ofrITF was synthesized and coupled to bovine serum albumin (BSA). Thisconjugate (and the unconjugated peptide) was used to raise polyclonalantibodies in rabbits. All procedures were standard protocols such asthose described in Ausubel et al. (supra). The anti-rITF antibodies wereused in an indirect immunoflouresce assay for visualization of rITF inrat tissues. Cryosections of rat tissues were prepared using standardtechniques, and fluorescein labelled goat anti-rabbit monoclonalantibody (labelled antibodies are available from such suppliersKirkegaard and Perry Laboratories, Gaithersberg, Md.; and Bioproductsfor Science, Inc., Indianapolis, Ind.) was used to detect binding ofrabbit anti-rITF antibodies. By this analysis rITF appears to be presentin the globlet cells of the small intestine but not in the stomach orthe pancreas.

[0050] Cloning of Human Intestinal Trefoil Factor

[0051] DNA encoding the rat intestinal trefoil factor can be used toidentify a cDNA clone encoding the human intestinal trefoil factor(hITF). This can be accomplished by screening a human colon cDNA librarywith a probe derived from rITF or with a probe derived from part of thehITF gene. The latter probe can be obtained from a human colon orintestinal cDNA using the polymerase chain reaction to isolate a part ofthe hITF gene. This probe can then serve as a specific probe for theidentification of clones encoding all of the hITF gene.

[0052] Construction of a cDNA Library

[0053] A human colon or intestinal cDNA library in λgtlO or λgtll, orsome other suitable vector is useful for isolation of hITF. Suchlibraries may be purchased (Clontech Laboratories, Palo Alto, Calif.:HLI034a, HLI0346b). Alternatively, a library can be produced usingmucosal scrapings from human colon or intestine. Briefly, total RNA isisolated from the tissue essentially as described by Chirgwin et al.(1979, Biochemistry 18:5294; see also Ausubel et al., supra). An oligo(dT) column is then used to isolate poly(A)⁺ RNA by the method of Avivet al. (1972, J. Mol. Biol. 134:743; see also Ausubel et al., supra).Double-stranded cDNA is then produced by reverse transcription usingoligo (dT)₁₂₋₁₈ or random hexamer primers (or both). RNAse H and E. coliDNA poll are then used to replace the RNA strand with a second DNAstrand. In a subsequent step E. coli DNA ligase and T4 DNA polymeraseare used to close gaps in the second DNA strand and create blunt ends.Generally, the cDNA created is next methylated with EcoRI methylase andEcoRI linkers are added (other linkers can be used depending on thevector to be used). In subsequent steps the excess linkers are removedby restriction digestion and the cDNA fragments are inserted into thedesired vector. See Ausubel et al., supra and Sambrook et al. (1990,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.) for detailed protocols. Useful vectorsinclude: λgtll, λgtlO, Lambda ZAP® II vector, Lambda Uni-ZAP™ XR vector,all available from Stratagene (La Jolla, Calif.).

[0054] The cDNA library must be packaged into phage; this is mostreadily accomplished by use of a commercial in vitro packaging kit,e.g., Gigapack® II Gold or Gigapack® II Plus (Stratagene, La Jolla,Calif.). See Ausubel et al. (supra) for packaging protocols and suitablehost strains. The library is preferably amplified soon after packaging;this step generates sufficient clones for multiple screening of thelibrary. See Ausubel et al. supra or Sambrook et al. supra for detailsof amplification protocols and procedures for storing the amplifiedlibrary.

[0055] Screening of the cDNA Library

[0056] To screen the library it must be placed on an appropriate hoststrain (e.g., Y1090 or Y1088 for λgtlO libraries, C600hflA for λgtlOlibraries). After plating the phage, plaques are transferred tonitrocellulose or nylon filters (See Ausubel et al., supra and Sambrooket al. supra). The filters are then probed with α³²P-labelled nicktranslated probe derived from rITF. The probe is preferentiallygenerated using a portion of the region of rITF DNA coding for thetrefoil structure (nucleotides 114 to 230 of SEQ ID NO.: 1, which encodecys32 to phe7l of SEQ ID NO.: 2). This region is conserved between rITF,pS2 and PSP, and it is likely that this region is conserved between rITFand hITF. Once a plaque is identified, several cycles of plaquepurification are required to isolate a pure clone encoding hITF. A phageDNA isolation is performed and the cDNA insert can be subcloned into anappropriate vector for restriction mapping and sequencing. If the phagevector is Lambda ZAP® II, coinfection with helper phage allows rescueand recircularization of pBluescript SK⁻ phagemid vector (Stratagene, LaJolla, Calif.) harboring the cDNA; alternatively the phage clone ispurified and the cDNA insert is subcloned into a vector suitable forrestriction mapping and sequencing. If the clone does not contain theentire hITF gene (as assessed by homology to rITF and the presence ofstart and stop codons), the library can be rescreened with the originalrITF probe or, preferably, with a probe generated from the hITF cloneobtained. If none of the clones contain the intact gene, it can bereconstructed from clones which bear overlapping fragments of hITF.

[0057] Direct Isolation of an HITF Probe by PCR

[0058] It is possible to isolate part of the hITF gene directly from thepackaged library or cDNA. To isolate a portion of hITF directly from thepackaged library, a pair of oligonucleotide primers and Taq polymeraseare used to amplify the DNA corresponding to the hITF gene. The primersused would be approximately 15-20 nucleotides long and correspond insequence to the 5′-most and 3′-most portions of the rITF codingsequence. Friedman et al. (in PCR Protocols: A Guide to Methods andApplications, Innis et al., Eds., Academic Press, San Diego) describe aprocedure for such amplification. Briefly, phage particles are disruptedby heating; Taq polymerase, primers (300 pmol of each), dNTPs, and Taqpolymerase buffer are added; and the mixture is thermally cycled toamplify DNA. The amplified DNA is isolated by agarose gelelectrophoresis. The ends of the fragment are prepared for ligation intoan appropriate vector by making them flush with T4 polymerase and, ifdesired, adding linkers. Alternatively, a restriction site may beengineered into the fragment by using primers which have sequence addedto their 5′ ends which sequence will generate an appropriate sticky endwhen digested. For example the sequence: 5′-GGGCGGCCGC-3′(SEQ ID NO.: 4)can be added to the 5′ end of each primer. This sequence includes theNotI restriction site flanked at the 5′ end by the sequence: GG. Theadditional nucleotides prevent the 5′ ends from denaturing andinterfering with subsequent restriction digestion with NotI. The gelpurified DNA of the appropriate size is next cloned into a cloningvector for sequencing and restriction mapping. This clone will not havethe entire hITF sequence, rather it will be a combination of hITF (theregion between the sequences corresponding to the primers) and rITF (the5′ and 3′ ends which correspond to the primer sequences). However, thisDNA can be used to generate a labelled probe (produced by nicktranslation or random primer labelling) which, since it is the correcthITF sequence, can be used in a high stringency screening of the libraryfrom which the cDNA was originally isolated. In an alternative approach,cDNA can be used in the above procedure instead of a packaged library.This eliminates the steps of modifying the cDNA for insertion into avector as well as cDNA packaging and library amplification. Ausubel etal. supra provides a protocol for amplification of a particular DNAfragment directly from cDNA and a protocol for amplification frompoly(A)⁺ RNA.

[0059] Identification of a Presumptive Human ITF Clone

[0060] A nick translated probe derived from rITF cDNA (corresponding tonucleotides 1 to 431 of SEQ ID NO.: 1) was used for Northern blotanalysis of poly(A)⁺ RNA derived from human intestinal mucosalscrapings. Probe hybridization and blot washing were carried outaccording to standard procedures. Probe (5×10⁵ cpm/ml hybridizationbuffer) was hybridized to the filter at 45 EC in 5×SSC with 30%formamide. The filter was then washed at 60 EC in 5×SSC with 40%formamide. Using this protocol a band was clearly visible after anovernight exposure of the filter with an intensifying screen. Thisresult indicated that there is sufficient homology between rITF and hITFto allow the use of probes derived from the sequence of the rITF genefor identification of the hITF gene.

[0061] A human intestinal cDNA library was obtained from Clontech (PaloAlto, Calif.). Alternatively, a human intestinal cDNA library may beproduced from mucosal scrapings as described above. Four oligonucleotideprobes were selected for screening the library cDNA. Two of the probescorrespond to sequences within the region of rITF encoding the trefoiland are referred to as internal probes (5′-GTACATTCTGTCTCTTGCAGA-3′ (SEQID NO.: 5) and 5′-TAACCCTGCTGCTGCTGGTCCTGG-3′ (SEQ ID NO.: 6)). Theother two probes recognize sequences within rITF but outside of thetrefoil encoding region and are referred to as external probes(5′-GTTTGCGTGCTGCCATGGAGA-3′ (SEQ ID NO.: 7) and5′-CCGCAATTAGAACAGCCTTGT-3′ (SEQ ID NO.: 8)). These probes were testedfor their utility by using them to screen the rat intestinal cDNAlibrary described above. Each of the four probes could be used toidentify a clone harboring all or part of the rITF gene. This resultindicates that these probes may be used to screen the human intestinallibrary for the presence of hITF.

[0062] The internal probes were used as described above to amplify a DNAfragment from human colon library cDNA (Clontech, Palo Alto, Calif.).Linkers were added to the isolated DNA fragment which was then insertedinto pBluescript phagemid vector (Stratagene, La Jolla, Calif.). Theregion of this clone corresponding to the sequence of human cDNA (i.e.,not including the sequence corresponding to the internal probes) wasused to make a radioactively labelled probe by randomoligonucleotide-primed synthesis (Ausbel et al., supra). This probe wasthen used to screen the human colon cDNA library. This screening led tothe identification of 29 clones. One of these clones (HuPCR-ITF) wasnick-translated to generate a probe for Northern analysis of poly(A)⁺RNA isolated from human intestinal mucosal scrapings. A single band ofroughly the same size as the rat transcript (approximately 0.45 kD) wasobserved.

[0063] Northern analysis of poly(A)⁺ isolated from human tissuesindicated that RNA corresponding to this probe was expressed in thesmall intestine and the large intestine but not in the stomach or theliver. These results indicate that the clone does not encode the humanhomolog of porcine PSP. Porcine PSP is expressed in porcine pancreas andis not significantly expressed in the small or large intestine. Theseresults also distinguish the cloned gene from pS2 which is expressed inthe stomach.

[0064]FIG. 6 shows the nucleic acid sequence information for human ITFcDNA, along with the deduced amino acid sequence in one-letter code (SEQID NO.: 3). This clone was obtained by the methods described above.

[0065] Production of hITF

[0066] The isolated hITF gene can be cloned into a mammalian expressionvector for protein expression. Appropriate vectors include pMAMneo(Clontech, Palo Alto, Calif.) which provides a RSV-LTR enhancer linkedto a dexamethasone-inducible MMTV-LTR promoter, an SV40 origin ofreplication (allows replication in COS cells), a neomycin gene, and SV40splicing and polyadenylation sites. This vector can be used to expressthe protein in COS cells, CHO cells, or mouse fibroblasts. The gene mayalso be cloned into a vector for expression in drosophila cells usingthe bacoluvirus expression system.

[0067] Purification of Intestinal Trefoil Factor

[0068] Intestinal trefoil factor can be purified from intestinal mucosalscrapings of human, rats or any other species which expresses ITF (pigsand cows may provide a source of ITF). The purification procedure usedfor PSP will be useful for the purification of ITF since the proteinsare likely to be homologous. Jorgensen et al. describes a method forpurification of PSP (1982, Regulatory Peptides 3:207). The preferredmethod is the second approach described by Jorgensen et al. (supra).This method involves chromatography of SP-Sephadex C-25 and QAE SephadexA-25 columns (Sigma, St. Louis, Mo.) in acidic buffer.

[0069] Anti-Intestinal Trefoil Factor Monoclonal Antibodies

[0070] Anti-intestinal trefoil factor monoclonal antibodies can beraised against synthetic peptides whose sequences are based on thededuced amino acid sequence of cloned hITF (SEQ ID NO.: 3). Mostcommonly the peptide is based on the amino-or carboxy-terminal 10-20amino acids of the protein of interest (here, hITF). The peptide isusually chemically cross-linked to a carrier molecule such as bovineserum albumin or keyhole limpet hemocyanin. The peptide is selected withthe goal of generating antibodies which will cross-react with the nativehITF. Accordingly, the peptide should correspond to an antigenic regionof the peptide of interest. This is accomplished by choosing a region ofthe protein which is (1) surface exposed, e.g., a hydrophobic region or(2) relatively flexible, e.g., a loop region or a â-turn region. In anycase, if the peptide is to be coupled to a carrier,it must have an aminoacid with a side chain capable of participating in the couplingreaction. See Hopp et al. (1983, Mol. Immunol. 20:483; 1982, J. Mol.Biol. 157:105) for a discussion of the issues involved in the selectionof antigenic peptides. A second consideration is the presence of aprotein homologous to hITF in the animal to be immunized. If such aprotein exists, it is important to select a region of hITF which is nothighly homologous to that homolog.

[0071] For hITF, peptides that correspond to the amino-terminal orcarboxy-terminal 15 amino acids are likely to be less homologous acrossspecies and exposed to the surface (and thus antigenic). Thus they arepreferred for the production of monoclonal antibodies. Purified hITF canalso be used for the generation of antibodies.

[0072] Genetic Disruption of a Trefoil Protein Impairs the Defense ofIntestinal Mucosa

[0073] As stated above, ITF is a member of the family of trefoilproteins that are expressed specifically and abundantly at the mucosalsurface of the gastrointestinal tract. Other members of this familyinclude pS2, which is expressed almost exclusively by foveolar cells ofthe stomach (Masiakowski et al., 1982, Nucl. Acids. Res. 10:7896;Jorgensen et al., 1982, Regulatory Peptides 3:231), and pancreaticspasmolytic peptide (SP), which is expressed by the pancreas and bygastric antrum (Jorgensen et al., supra). As described above, theexpression of these proteins is enhanced in the proximity of the injuredbowel.

[0074] In order to study the role of ITF in vivo, the gene was renderednon-functional by targeted disruption in mice.

[0075] Isolation of the Murine ITF Gene and Generation of ITF-DeficientMice

[0076] The murine ITF gene was isolated from a phage genomic libraryusing the rat ITF cDNA sequence as a probe, and its identity wasconfirmed by nucleotide sequencing using standard techniques (Mashimo etal., 1995, Biochem. Biophys. Res. Comm. 210:31).

[0077] A targeting vector for disrupting the gene by homologousrecombination in embryonic stem (ES) cells was designed and constructed,as shown in FIG. 7. The entire second exon (Ex2) of the murine ITF gene,which is contained within the XbaI-EcoRI fragment shown, was replacedwith the neomycin resistance (neo) gene cassette. As the deletedsequence encodes most of the “trefoil domain”, the ability of anyresultant peptides to produce the looping structure characteristic oftrefoil proteins is abolished. A positive-negative selection strategy(Mansour et al., 1988, Nature 336:348) was used to enrich for homologousrecombination events in the embryonic stem (ES) cells by selecting forneo within the homologous DNA and against a herpes simplex virusthymidine kinase gene (hsv-tk) placed at the 3′ end of the targetingvector. The pPNT plasmid (Tybulewicz et al., 1991, Cell 65:1153) wasused to construct the targeting vector. The targeting vector waslinearized with the restriction enzyme NotI and electroporated intopluripotent J1 ES cells (Li et al., 1992, Cell 69:915) under conditionspreviously described (Strittmatter et al., 1995, Cell 80:445).Disruption of the ITF gene in ES cells following homologousrecombination was distinguished from random integration of the targetingvector by Southern blot analysis of genomic DNA from individual clonesof cells digested with the restriction enzyme XhoI. The pITF2 probeidentified a 19 kb “wild type” fragment and a 23 kb “knock out” fragmentcreated by introduction of an XhoI site upon homologous insertion of thetargeting vector. Approximately 10% of neomycin-resistant ES clones werefound to have undergone homologous ITF recombination using this method.

[0078] The polymerase chain reaction (PCR) was used to confirm thetargeted mutation as follows. A 200 bp region of DNA was amplified usingprimers spanning exon 2 of ITF (5′-GCAGTGTAACAACCGTGGTTGCTGC-3′ (SEQ IDNO.: 9) and 5′-TGACCCTGTGTCATCACCCTGGC-3′ (SEQ ID NO.: 10)); and a 400bp region of the neo gene was amplified with a second set of primers(5′-CGGCTGCTCTGATGGCCGCC-3′ (SEQ ID NO.: 11) and5′-GCCGGCCACAGTCGATGAATC-3′ (SEQ ID NO.: 12)) The DNA template for thePCR reaction was obtained from tail tissue. Approximately 0.5 cm of thetail was cut off each animal, and the samples were digested withproteinase-K (200 μl at 0.5 mg/ml in 50 mM Tris-HCl pH 8.0 and 0.5%Triton X-100; Sigma, St. Louis, Mo.) at 55° C. overnight. One μl of thismixture was added directly to a 25 μl PCR reaction (per Stratagene,Menosha, Wis.). The reaction was begun with a “hot start” (incubation at96° C. for 10 minutes), and the following cycle was repeated 30 times:72° C. for 120 seconds (hybridization and elongation) and 96° C. for 30seconds (denaturation). Ten μl of each reaction mixture waselectrophoresed on a 2% agarose gel. Wild type animals were identifiedby the presence of a 200 bp fragment, corresponding to an intact ITFgene, heterozygous animals were identified by the presence of this bandand, in addition, a 400 bp fragment produced by amplification of the neogene, and ITF-deficient (knock out) animals were identified by thepresence of only the fragment corresponding to the neo gene.

[0079] Two ES clones, which arose independently, were used to derive twolines of mice lacking ITF. These mice were screened by Southern genomicblot analysis as described for ES clones, or by PCR.

[0080] Analysis of Trefoil Peptide Expression in Wild Type and MutantMice

[0081] Although expression of ITF is abolished in the mutant mice,expression of other trefoil genes is preserved. Northern blot analysiswas performed using cDNA probes for ITF (Suemori et al., 1991, Proc.Natl. Acad. Sci. USA 88:11017), SP (Jeffrey et al., 1994Gastroenterology 106:336), and, as a positive control, glyceraldehyde3-phosphate dehydrogenase (GAPDH). The nucleic acid probe for murine pS2was made by reverse transcription-polymerase chain reaction (RT-PCR)using the oligonucleotide pairs: 5′-GAGAGGTTGCTGTTTTGATGACA-3′ (SEQ IDNO.: 13) and 5′-GCCAAGTCTTGATGTAGCCAGTT-3″ (SEQ ID NO.: 14), which weresynthesized based on the published mouse pS2 cDNA sequence (GenBankAccession Number: Z21858). The GeneAmp RNA PCR Kit (Perkin Elmer) wasused according to the manufacturer's instructions, as was the PCR™II(Invitrogen) cloning vector. RNA was extracted from the followingtissues from both wild type and ITF-deficient (knock out) mice: stomach,duodenum, terminal ileum, right colon, appendix, transverse colon, leftcolon, and rectum. Fifteen μg of total RNA from each sample wereelectrophoresed on a 1% agarose gel, and transferred to nitrocellulosepaper. Following hybridization, washing, and autoradiography, wild typemice exhibited a pattern of tissue expression considered normal: ITF wasexpressed in the small intestine and colon, which is the same expressionpattern seen for ITF in the rat and human. The analysis of mutant miceconfirmed the lack of ITF expression in the gastrointestinal tract. Incontrast, the expression of the other trefoil proteins, SP and pS2, areunaltered in the gastrointestinal tract of mutant mice. SP was expressedin the stomach and, at lower levels, in the duodenum of both wild typeand mutant mice. Similarly, pS2 was expressed in the stomach of bothwild type and ITF-deficient mice.

[0082] Immunocytochemistry Reveals that ITF is not Expressed in theColon of ITF-deficient Mice

[0083] In order to confirm that ITF protein was not expressed by ITFknock out mice, immunocytochemistry was performed as follows. Tissuefrom the colon and small intestine was fixed in the course of perfusion,immersed in 4% paraformaldehyde (McLean et al., 1974, J. Histochem.Cytochem. 22:1077), and embedded in paraffin. Sections were collectedand stained either with a polyclonal antibody raised against a syntheticpeptide from the predicted 18 carboxy-terminal amino acids of murine ITFor a monoclonal antibody against colonic mucin (Podolsky et al., 1986,J. Clin. Invest. 77:1263). Primary antibody binding was visualized witha biotinylated secondary antibody, Avidin DH, biotinylated horseradishperoxidase H, and diaminobenzidine tetrahydrochloride reagents accordingto the manufacturer's instructions (VectaStain ABC, Vector Laboratories,Bulingame, Calif.). Following immunocytochemistry, the sections werecounterstained with hematoxylin and viewed. Goblet cells in the colon ofwild type mice were immunoreactive with both antibodies, stainingpositively for ITF and mucin. In contrast, the goblet cells in the colonof ITF-deficient mice lacked detectable ITF but continued to expresscolonic mucin.

[0084] Induction of Mild Colonic Epithelial Injury with Dextran SulfateSodium

[0085] ITF-deficient mice derived from each ES clone appear to developnormally and are grossly indistinguishable from heterozygous and wildtype litter mates. Their growth is not retarded and they reach maturitywithout evident diarrhea or occult fecal blood loss. However, the colonof ITF-deficient mice may be more prone to injury than the colon, ofwild type mice. To investigate this hypothesis, dextran sulfate sodium(DSS), which reproducibly creates mild colonic epithelial injury withulceration in mice (Kim et al., 1992, Scand. J. Gastroent. 27:529; Wellset al., 1990, J. Acquired Immune Deficiency Syndrome 3:361; Okayasu etal., 1990, Gastroenterology 98:694) was administered in the animals'drinking water. After standardization of DSS effects in comparable wildtype mice, a group of 20 wild type and 20 ITF-deficient mice (littermates from heterozygous crosses, weighing >20 grams each) were treatedwith 2.5% DSS in their drinking water for nine days.

[0086] Although 85% of wild type mice and 100% of ITF-deficient micetreated with DSS demonstrate occult blood (using Hemoccult, Smith KlineDiagnostics, San Jose, Calif.) in their stool during the period oftreatment, ITF-deficient mice were markedly more sensitive to theinjurious effects of DSS. Fifty percent of ITF-deficient mice developedfrankly bloody diarrhea and died (FIG. 8). In contrast, only 10% of wildtype mice treated similarly exhibited bloody diarrhea, and only 5% died.Weight loss was also significantly more pronounced in ITF-deficient micethan wild type mice receiving DSS.

[0087] ITF-Deficient Mice Treated with Dextran Sulfate Sodium (DSS)Develop Severe Colonic Erosions

[0088] After seven days of treatment with DSS (2.5% w/v), the colons ofwild type and ITF-deficient mice were examined histologically. Leftcolon transections were fixed in 4% paraformaldehyde, mounted inparaffin, and stained with hematoxylin and eosin. Multiple sites ofobvious ulceration and hemorrhage were present in the colon ofITF-deficient mice, while the colons of most wild type mice were grosslyindistinguishable from those of untreated mice. Histological examinationof the DSS-treated ITF-deficient colon confirmed the presence ofmultiple erosions and intense inflammatory changes including cryptabscesses. Damage was more pronounced in the distal colon, i.e., thedescending colon, sigmoid colon, and rectum, which contained large,broad areas of mucosal ulceration. When similarly inspected, mucosalerosions could be seen in the tissue of 80% of the DSS-treated wild typemice, but most were small lesions that also appeared to be healing, withcomplete re-epithelialization of most lesions. There was no evidence ofre-epithelialization in the colons of ITF-deficient mice exposed to DSS.

[0089] During the normal course of growth and development, intestinalepithelial cells originate from stem cells in the intestinal crypts andrapidly progress up the crypt and villus to be extruded from the villustip within five days. After intestinal injury, the epithelial coveringis repopulated by cells which appear to generate signals to heal thelesion by modulation of epithelial and mesenchymal cell growth andmatrix formation (Poulsom et al., 1993, J. Clin. Gastroenterol. 17:S78).In vitro evidence suggests that trefoil proteins play a key role inre-establishing mucosal integrity after injury. Despite the normalrestriction of SP and pS2 expression to the proximal gastrointestinaltract, these trefoil proteins and ITF are abundantly expressed at sitesof colonic injury and repair.

[0090] The DSS model described above provides a system for testing theprotective effects of ITF, other trefoil peptides, or active polypeptidefragments or variants thereof. One can administer a molecule to betested to DSS-treated mice, either wild type or ITF-deficient mice, anddetermine whether the molecule has therapeutic effects by performing theassays described above.

[0091] In addition to the use of DSS, any chemical compound that isknown to damage the mucosa lining the digestive tract can be used toassay the proteins of the invention. These compounds include, but arenot limited to, alcohol, indomethacin, and methotrexate. For example,methotrexate (MTX) can be administered intraperiotoneally to mice at adose of 40 mg/kg. One group of MTX-treated animals could be given, inaddition, the protein in question. Various parameters, such as bodyweight, the presence of lesions in the digestive tract, and mortality ofthese animals could then be compared to equivalent measurements takenfrom animals that were not treated with the protein.

[0092] In Situ H. pylori Binding Assay

[0093] One method for determining whether a given protein (or proteinfragment or variant) is useful in the prevention or treatment ofdiseases associated with H. pylori infection is to examine it in thecontext of an established animal model of H. pylori infection. One suchmodel was recently developed by Falk et al. (1995, Proc. Natl. Acad.Sci. USA 92:1515-1519). This model involves the use of transgenic micethat express the enzyme α-1,3/4-fucosyltransferase and, as aconsequence, express Le^(b) on the surface of mucosal cells that boundclinical isolates of H. pylori. If the addition of a protein, such asITF, to this system reduces the level of H. pylori binding to themucosal cell, the protein would be considered an inhibitor of H. pylori.More specifically, the assay could be carried out as follows. H. pyloriare obtained, for example, from patients with gastric ulcers or chronicactive gastritis, grown to stationary phase, and labeled, for examplewith digoxigenin or fluorescein isothiocyanate (FITC). The labeledbacteria are then exposed, together with the protein of interest, tofrozen sections prepared from the stomach, duodenum, ileum, or liver ofadult transgenic mice (as described above). As a control, the experimentcould be performed in parallel using tissue from a wild type littermate.The sections are fixed with ice-cold methanol for 5 minutes, rinsedthree times with wash buffer (TBS; 0.1 mM CaCl₂, 1 mM MnCl₂, 1 MM MgCl₂;10 minutes/cycle), and treated with blocking buffer (BoehringerMannheim; see also Falk supra). Bacteria are diluted to an OD₆₀₀ of 0.05with dilution buffer (TBS; 0.1 mM CaCl₂, 1 mM MnCl₂, 1 mM MgCl₂containing leupeptin (1 μg/ml), aprotinin (1 μg/ml),[-1-p-tosylamido-2-phenylethyl chloromethyl ketone (100 μg/ml),phenylmethylsulfonyl fluoride (100 ig/ml), and pepstatin A (1 μg/ml))and overlaid on the sections for 2 hours at room temperature in ahumidified chamber. Slides are then washed six times in wash buffer on arotating platform (5 minutes/cycle at room temperature).Digoxigenin-labeled bacteria are visualized on washed slides withFITC-conjugated sheep anti-digoxigenin immunoglobulin (BoehringerMannheim) diluted 1:100 in histoblocking buffer. Nuclei were stainedwith bisbenzimide (Sigma). For blocking controls, digoxigenin-conjugatedstationary-phase bacteria can be suspended in dilution buffer to anOD₆₀₀ of 0.05 and shaken with or without Le^(b)-HSA or Le^(a)-HSA (finalconcentration, 50 μg/ml; reaction mixture, 200 μl) for 1 hour at roomtemperature. The suspension is then overlaid on methanol-fixed frozensections.

[0094] Use

[0095] In the practice of the present invention, ITF may be administeredorally, intravenously, or intraperitoneally for treatment of pepticulcer diseases, inflammatory bowel diseases, and for protection of theintestinal tract from injury caused by bacterial infection, radiationinjury or other insults. The mode of administration, dosage, andformulation of ITF will depend upon the condition being treated.

[0096] Skilled pharmacologists are able to readily determine appropriatedosage regimens. As trefoil peptides are not degraded within thedigestive tract, it is expected that the route of administration will beoral, and that the dosage will range from 1 to 500 mg, taken once tothree times per day. The peptide could be administered, for example, inthe form of a tablet, capsule, or pill, or could be suspended in asolution, such as a syrup, that the patient swallows. Alternatively, thesolution containing the peptide may be administered as a gastric lavage.The peptide may also be included in a solution that is administered asan enema, or it may be administered as a suppository.

[0097] Deposit Statement

[0098] The human intestinal trefoil clone described herein has beendeposited under conditions in which access will be available during thependency of the present patent application to those determined by theCommissioner of Patents and Trademarks to be entitled thereto under 37C.F.R. § 1.14 and 35 U.S.C. § 122. More specifically, the humanintestinal trefoil clone described herein has been deposited with theAmerican Type Culture Collection (Manassas, Va.) and assigned AccessionNumber 98767.

[0099] Other Embodiments

[0100] ITF may be used to produce monoclonal antibodies for thedetection of ITF in intestinal tissue or blood serum by means of anindirect immunoassay. ITF may be detectably labelled and used in an insitu hybridization assay for the detection of ITF binding sites. Labelsmay include, but are not limited to, fluorescein or a radioactiveligand.

[0101] ITF may be used to protect and stabilize other proteins. Thisprotection is accomplished by forming a hybrid molecule in which all orpart of ITF is fused to either the carboxy-terminus or theamino-terminus (or both) of the protein of interest. Because ITF isresistant to degradation in the digestive system, it will protect theprotein of interest from such degradation. As a consequence, the proteinof interest is likely to remain active in the digestive system and/orwill be more readily absorbed in an intact form.

[0102] Trefoil proteins, including ITF, can be used to promote healingor prevent wounding of corneal tissue.

[0103] Stably dimerized trefoil protein can be used in the methods ofthe invention. Such molecules can be prepared by stably crosslinkingmonomers of trefoil or by expressing a gene encoding a tandem repeat ofa trefoil protein (e.g., ITF) or a portion thereof (e.g., a portioncapable of forming the three loop structure characteristic of trefoilproteins).

[0104] Also useful in the method of the invention are trefoil proteinsproduced by chemical synthesis.

[0105] Trefoil proteins can be used to treat other disorders, e.g.,Crohn's disease.

[0106] Other embodiments are within the following claims.

1 20 431 base pairs nucleic acid single linear Coding Sequence 18...2601 GAAGTTTGCG TGCTGCC ATG GAG ACC AGA GCC TTC TGG ATA ACC CTG CTG 50 MetGlu Thr Arg Ala Phe Trp Ile Thr Leu Leu 1 5 10 CTG GTC CTG GTT GCT GGGTCC TCC TGC AAA GCC CAG GAA TTT GTT GGC 98 Leu Val Leu Val Ala Gly SerSer Cys Lys Ala Gln Glu Phe Val Gly 15 20 25 CTA TCT CCA AGC CAA TGT ATGGCG CCA ACA AAT GTC AGG GTG GAC TGT 146 Leu Ser Pro Ser Gln Cys Met AlaPro Thr Asn Val Arg Val Asp Cys 30 35 40 AAC TAC CCC ACT GTC ACA TCA GAGCAG TGT AAC AAC CGT GGT TGC TGT 194 Asn Tyr Pro Thr Val Thr Ser Glu GlnCys Asn Asn Arg Gly Cys Cys 45 50 55 TTT GAC TCC AGC ATC CCA AAT GTG CCCTGG TGC TTC AAA CCT CTG CAA 242 Phe Asp Ser Ser Ile Pro Asn Val Pro TrpCys Phe Lys Pro Leu Gln 60 65 70 75 GAG ACA GAA TGT ACA TTT TGAAGCTGTCCAGGCTCCAG GAAGGGAGCT CCACAC 298 Glu Thr Glu Cys Thr Phe 80 TGGACTCTTGCTGATGGTAG TGGCCCAGGG TAACACTCAC CCCTGATCTG CTCCCTCG 358 CCGGCCAATATAGGAGCTGG GAGTCCAGAA GAATAAAGAC CTTACAGTCA GCACAAGG 418 GTTCTAATTG CGG431 81 amino acids amino acid linear protein internal 2 Met Glu Thr ArgAla Phe Trp Ile Thr Leu Leu Leu Val Leu Val Ala 1 5 10 15 Gly Ser SerCys Lys Ala Gln Glu Phe Val Gly Leu Ser Pro Ser Gln 20 25 30 Cys Met AlaPro Thr Asn Val Arg Val Asp Cys Asn Tyr Pro Thr Val 35 40 45 Thr Ser GluGln Cys Asn Asn Arg Gly Cys Cys Phe Asp Ser Ser Ile 50 55 60 Pro Asn ValPro Trp Cys Phe Lys Pro Leu Gln Glu Thr Glu Cys Thr 65 70 75 80 Phe 400base pairs nucleic acid single linear Coding Sequence 2...223 3 G ATGCTG GGG CTG GTC CTG GCC TTG CTG TCC TCC AGC TCT GCT GAG GA 49 Met LeuGly Leu Val Leu Ala Leu Leu Ser Ser Ser Ser Ala Glu Glu 1 5 10 15 TACGTG GGC CTG TCT GCA AAC CAG TGT GCC GTG CCG GCC AAG GAC AGG 97 Tyr ValGly Leu Ser Ala Asn Gln Cys Ala Val Pro Ala Lys Asp Arg 20 25 30 GTG GACTGC GGC TAC CCC CAT GTC ACC CCC AAG GAG TGC AAC AAC CGG 145 Val Asp CysGly Tyr Pro His Val Thr Pro Lys Glu Cys Asn Asn Arg 35 40 45 GGC TGC TGCTTT GAC TCC AGG ATC CCT GGA GTG CCT TGG TGT TTC AAG 193 Gly Cys Cys PheAsp Ser Arg Ile Pro Gly Val Pro Trp Cys Phe Lys 50 55 60 CCC CTG CAG GAAGCA GAA TGC ACC TTC TGAGGCACCT CCAGCTGCCC CTG 243 Pro Leu Gln Glu AlaGlu Cys Thr Phe 65 70 GGATGCAGGC TGAGCACCCT TGCCCGGCTG TGATTGCTGCCAGGCACTGT TCATCTCA 303 TTTTCTGTCC CTTTGCTCCC GGCAAGCTTT CTGCTGAAAGTTCATATCTG GAGCCTGA 363 TCTTAACGAA TAAAGGTCCC ATGCTCCACC CGAAAAA 400 10base pairs nucleic acid single linear 4 GGGCGGCCGC 10 21 base pairsnucleic acid single linear 5 GTACATTCTG TCTCTTGCAG A 21 24 base pairsnucleic acid single linear 6 TAACCCTGCT GCTGCTGGTC CTGG 24 21 base pairsnucleic acid single linear 7 GTTTGCGTGC TGCCATGGAG A 21 21 base pairsnucleic acid single linear 8 CCGCAATTAG AACAGCCTTG T 21 25 base pairsnucleic acid single linear 9 GCAGTGTAAC AACCGTGGTT GCTGC 25 23 basepairs nucleic acid single linear 10 TGACCCTGTG TCATCACCCT GGC 23 20 basepairs nucleic acid single linear 11 CGGCTGCTCT GATGGCCGCC 20 21 basepairs nucleic acid single linear 12 GCCGGCCACA GTCGATGAAT C 21 23 basepairs nucleic acid single linear 13 GAGAGGTTGC TGTTTTGATG ACA 23 23 basepairs nucleic acid single linear 14 GCCAAGTCTT GATGTAGCCA GTT 23 60amino acids amino acid linear protein 15 Glu Ala Gln Thr Glu Thr Cys ThrVal Ala Pro Arg Glu Arg Gln Asn 1 5 10 15 Cys Gly Phe Pro Gly Val ThrPro Ser Gln Cys Ala Asn Lys Gly Cys 20 25 30 Cys Phe Asp Asp Thr Val ArgGly Val Pro Trp Cys Phe Tyr Pro Asn 35 40 45 Thr Ile Asp Val Pro Pro GluGlu Glu Cys Glu Phe 50 55 60 62 amino acids amino acid linear protein 16Glu Lys Pro Ala Ala Cys Arg Cys Ser Arg Gln Asp Pro Lys Asn Arg 1 5 1015 Val Asn Cys Gly Phe Pro Gly Ile Thr Ser Asp Gln Cys Phe Thr Ser 20 2530 Gly Cys Cys Phe Asp Ser Gln Val Pro Gly Val Pro Trp Cys Phe Lys 35 4045 Pro Leu Pro Ala Gln Glu Ser Glu Glu Cys Val Met Glu Val 50 55 60 59amino acids amino acid linear protein 17 Gln Glu Phe Val Gly Leu Ser ProSer Gln Cys Met Ala Pro Thr Asn 1 5 10 15 Val Arg Val Asp Cys Asn TyrPro Thr Val Thr Ser Glu Gln Cys Asn 20 25 30 Asn Arg Gly Cys Cys Phe AspSer Ser Ile Pro Asn Val Pro Trp Cys 35 40 45 Phe Lys Pro Leu Gln Glu ThrGlu Cys Thr Phe 50 55 73 amino acids amino acid linear protein internal18 Met Leu Gly Leu Val Leu Ala Leu Leu Ser Ser Ser Ser Ala Glu Glu 1 510 15 Tyr Val Gly Leu Ser Ala Asn Gln Cys Ala Val Pro Ala Lys Asp Arg 2025 30 Val Asp Cys Gly Tyr Pro His Val Thr Pro Lys Glu Cys Asn Asn Arg 3540 45 Gly Cys Cys Phe Asp Ser Arg Ile Pro Gly Val Pro Trp Cys Phe Lys 5055 60 Pro Leu Gln Glu Ala Glu Cys Thr Phe 65 70 60 amino acids aminoacid linear protein 19 Glu Ala Gln Thr Glu Thr Cys Thr Val Ala Pro ArgGlu Arg Gln Asn 1 5 10 15 Cys Gly Phe Pro Gly Val Thr Pro Ser Gln CysAla Asn Lys Gly Cys 20 25 30 Cys Phe Asp Asp Thr Val Arg Gly Val Pro TrpCys Phe Tyr Pro Asn 35 40 45 Thr Ile Asp Val Pro Pro Glu Glu Glu Cys GluPhe 50 55 60 106 amino acids amino acid linear protein 20 Glu Lys ProAla Ala Cys Arg Cys Ser Arg Gln Asp Pro Lys Asn Arg 1 5 10 15 Val AsnCys Gly Phe Pro Gly Ile Thr Ser Asp Gln Cys Phe Thr Ser 20 25 30 Gly CysCys Phe Asp Ser Gln Val Pro Gly Val Pro Trp Cys Phe Lys 35 40 45 Pro LeuPro Ala Gln Glu Ser Glu Glu Cys Val Met Gln Val Ser Ala 50 55 60 Arg LysAsn Cys Gly Tyr Pro Gly Ile Ser Pro Glu Asp Cys Ala Ala 65 70 75 80 ArgAsn Cys Cys Phe Ser Asp Thr Ile Pro Glu Val Pro Trp Cys Phe 85 90 95 PhePro Met Ser Val Glu Asp Cys His Tyr 100 105

1. A substantially pure and biologically active fragment of thepolypeptide encoded by the cDNA clone deposited with the American TypeCulture Collection and assigned Accession Number
 98767. 2. The fragmentof claim 1, wherein said fragment comprises a trefoil structure.
 3. Thefragment of claim 1, wherein said fragment is at least 40 contiguousamino acids in length.
 4. The fragment of claim 3, wherein said fragmentis at least 50 contiguous amino acids in length.
 5. The fragment ofclaim 4, wherein said fragment is at least 60 contiguous amino acids inlength.
 6. A therapeutic composition comprising the fragment of claim 1and a pharmacologically acceptable carrier.
 7. A method for treating adigestive disorder in a human patient, said method comprisingadministering to said patient the therapeutic composition of claim
 6. 8.The method of claim 7, wherein said digestive disorder is non-ulcerdyspepsia, gastritis, peptic ulcer, duodenal ulcer, gastro-espohagealreflux disease, or Crohn's Disease.
 9. A method for treating a lesion ofthe alimentary canal in a human patient, said method comprisingadministering to said patient the therapeutic composition of claim 6.10. The method of claim 9, wherein said lesion is in the mucosa of thealimentary canal.
 11. The method of claim 9, wherein said lesion is inthe mouth of said patient.
 12. The method of claim 9, wherein saidlesion is in the esophagus of said patient.
 13. The method of claim 9,wherein said lesion is in the stomach of said patient.
 14. The method ofclaim 9, wherein said lesion is in the intestine of said patient. 15.The method of claim 9, wherein said patient is receiving radiationtherapy or chemotherapy for the treatment of cancer.
 16. The method ofclaim 9, wherein said patient is receiving a drug that damages thealimentary canal.
 17. The method of claim 9, wherein said administrationis oral.
 18. The method of claim 9, wherein said administration furthercomprises administration of mucin glycoprotein preparations.